Pharmaceutical composition comprising (S)-N-ethyl-3-[1-dimethylamino)ethyl]-N-methyl-phenyl-carbamate in free base or acid addition salt form and an anti-oxidant. Said pharmaceutical compositions may be delivered to a patient using a transdermal delivery device.

This invention relates to a pharmaceutical composition for systemic administration of a phenyl carbamate, e.g. by transdermal administration. In particular this invention relates to a pharmaceutical composition of the phenyl carbamatexe2x80x94(S)-N-ethyl-3-[1-dimethylamino)ethyl]-N-methyl-phenyl-carbamatexe2x80x94(hereinafter referred to as compound A) in free base or acid addition salt form as disclosed in published UK patent application GB 2 203 040, the contents of which are incorporated herein by reference.
Compound A is useful in inhibiting acetylcholinesterase in the central nervous system, e.g. for the treatment of Alzheimer""s disease.
A transdermal composition in the form of a patch is described in Example 2 of GB 2,203,040 according to which compound A is mixed with two polymers and a plasticiser to form a viscous mass. This mass is applied to a foil which is cut into patches.
It has now been found after exhaustive testing that compound A is susceptible to degradation, particularly in the presence of oxygen. The transdermal composition described in GB 2203040 has been found to degrade, possibly by oxidative degradation, despite the formation of an occlusive polymer matrix around compound A and its storage in air-tight packaging.
The present applicant has found that stable pharmaceutical compositions comprising compound A can now be obtained, which show insignificant degradation of compound A over a prolonged time period, e.g. 2 years, as indicated by standard tests, e.g. stress tests.
In one aspect, the invention provides a pharmaceutical composition comprising Compound A in free base or acid addition salt form and an anti-oxidant.
The pharmaceutical compositions of the present invention show a reduction in degradation by-products in stress stability tests.
The pharmaceutical compositions of the invention may contain high amounts of compound A, e.g. from 1 to 40% by weight, e.g. 10-35%, more particularly 20-35%, e.g. 30%.
The compound A may be in any of a wide variety of pharmaceutical diluents and carriers known in the art. The diluent or carrier may contain trace amounts of free radicals without affecting the stability of the pharmaceutical composition of the invention.
The diluent or carrier is preferably one or more polymers, more preferably a hydrophilic polymer or polymers. In a preferred embodiment the diluent of carrier is selected from at least one polymer selected from acrylate polymers, and polymethacrylate polymers. The polymers preferably have a mean molecular weight of from about 50,000 to about 300,000 Daltons, e.g. 100,000 to 200,000 Daltons. The polymers preferably are capable of forming a film, thus to be compatible to the skin.
As a polymer one can mention in particular an acrylate co-polymer, e.g. co-polymers of butyl acrylate, ethyl hexyl acrylate and vinyl acetate. Preferably the polymer is cross-linked. A preferred acrylate polymer is one of the Durotak brand available from National Starch and Chemical Company, Zutphen, Holland, e.g. Durotak 87-2353 (hereinafter polymer A), 387-2051 or 387-2052 (hereinafter polymer D).
The diluent or carrier is preferably present in an amount of up to 90%, more preferably 70% by weight base on the total weight of the pharmaceutical composition.
The polymer, when a hydrophilic polymer, may conveniently take up water and is permeable to water, e.g. moisture from the skin, although the polymer may be insoluble in water. The polymer may swell and provide release of a large amount of pharmacologically active agent leading to a high concentration gradient of pharmacologically active agent between the skin surface and stratum comeum at a pH of from 4 to 7, preferably at skin pH, e.g. around 5.5. If desired such polymers may be soluble in organic solvents.
Examples of suitable polymers include polyacrylamide and its co-polymers, polyvinylpyrrolidone (PVP), vinyl acetate/vinyl alcohol co-polymers, polyvinyl alcohol (PVA) and derivatives, ethyl cellulose and other cellulose and starch derivatives.
Hydrophilic polyacrylates are preferred polymers. The polyacrylate may be substituted, e.g. a methacrylate. They may be commercially available acrylate/methacrylate co-polymers. Some or all of the acid groups may be esterified, e.g. with alkyl (C1-10) groups, more particularly alkyl groups having 1 to 4 carbon atoms such as methyl or ethyl groups.
Examples of commercially available polymers of this type include:
1) Polymers of methacrylate containing alkyl (C1-4) ester groups. Preferably the polymer matrix is a mixture of an acrylate polymer and a methacrylate polymer e.g. in a weight ratio of from 5:1 to 1:1, e.g. 4:1 to 2:1 e.g. 3:1, e.g. butylmethylacrylate and methylmethylacrylate. MW 20000, e.g. Plastoid B from Rxc3x6hm, Darmstadt, Germany (hereinafter polymer B).
2) Polymers of acrylate and methacrylate esters containing methyl and ethyl neutral ester groups and trimethylaminoethyl cationic ester groups. Chloride ions may be present. Mean Molecular weight 150000 Daltons. Viscosity (20xc2x0 C.), maximum 15 cP. Refractive index 1.380-1.385. Density 0.815-0.835 g/cm3. Ratio of cationic ester groups to neutral alkyl groups 1:20 giving an alkali count of 28.1 mg KOH per gram polymer (Eudragit RL 100 Registered Trade Mark available from Rxc3x6hm) or 1:40 giving an alkali count of 15.2 mg KOH per gram polymer (Eudragit RS 100 Registered Trade Mark, also available from Rxc3x6hm).
3) Polymers of methacrylate esters containing trimethylaminoethyl cationic ester groups and other neutral (C1-4)alkyl ester groups. Chloride ions may be present. Mean molecular weight 150,000. Viscosity (20xc2x0 C.) 10 cP. Refractive Index 1.38. Density 0.815. Alkali number of 180 mg KOH per gram polymer (Eudragit E 100, Registered Trade Mark, also available from Rxc3x6hm and hereinafter referred to a polymer C).
If desired the pharmaceutical composition may contain other additives, such as plasticizers and/or softeners preferably skin compatible tensides, e.g. to provide flexibility to the pharmaceutical composition, and/or to dissolve partially or totally compound A.
Examples of additives include:
1) Polyoxyethylene fatty alcohol ethers. The alcohol may e.g. be a C12-18 alcohol. The HLB value may be e.g. from 10 to 18. A preferred example is polyoxyethylene-(10) oleyl ether. A suitable ether may have a viscosity (25xc2x0 C.) of about 100 cP, a solidification point of about 16xc2x0 C., an HLB value of 12.4 and an acid count maximum 1.0 (Brij 97 Registered Trade Mark available from Atlas Chemie, Germany).
2) Polyoxyethylene Sorbitan fatty acid esters. The fatty acid may be e.g. a C12-18 fatty acid. The HLB value may be e.g. from 10 to 18. A preferred example is polyoxyethylene-(20) sorbitan monooleate, e.g. Tween 80, Registered Trade Mark available from Atlas Chemie, Germany.
3) Polyoxyethylene-(5-40) stearic acid esters, e.g. Myrj (Registered Trade Mark) available from Atlas Chemie, Germany.
4) Polyoxyethylene glycol fatty alcohol ethers, e.g. polyethylene glycol-(6-25) cetyl ether, glycerin polyethylene ricinoleate, glycerin polyethylene glycol stearate (Cremophor brand, Registered Trade Mark available from BASF Germany).
5) Polyoxyethylene glycols of MW from 200 to 600 Daltons, e.g. 300 or 400 Daltons.
6) Esters of poly(2-7)ethylene glycol glycerol ether having at least one hydroxyl group and an aliphatic (C6-22) carboxylic acid, e.g. Polyethylene glycol-(7) glyceryl cocoate, e.g. Cetiol HE, Registered Trade Mark, from Henkel, Germany.
7) Adipic acid lower alkyl esters, e.g. di-n-butyl adipate and diisopropyl adipate.
8) Glycerin polyethylene glycol ricinoleate, e.g. Product of 35 moles ethylene oxide and castor oil, e.g. Brand Cremophor EL Registered Trade Mark, obtainable from BASF, Germany.
9) Tracetin-(1,2,3).
10) Fatty acid, e.g. a C12-18 fatty acid.
11) Fatty alcohol, e.g. a C12-18 fatty alcohol.
The amount and type of additive required may depend on a number of factors, e.g. the HLB value of the tenside and the flexibility of the pharmaceutical required. The amount of additive does not significantly influence the capability of the polyacrylate to form films. Generally the weight ratio of tenside to the polymer may be from about 1:10 to 5:1, e.g. 1:10 to 1:3.
Preferably, however, no such additive is present or is only present in an amount less than 1% by weight based on the total weight of the pharmaceutical composition.
The pharmaceutical composition may contain skin penetration promoters, e.g. 1-dodecylazacycloheptan-2-one(azone) and N,N-diethyl-m-toluamide (DEET).
The amount and type of skin penetration promoter, and/or additives present may depend on a number of factors. Generally the weight ratio of skin penetration promoting agent to hydrophilic polymer will be from about 1:1 to 1:10. Preferably the amount of tenside and/or skin penetration promoter may be from about 3 to about 50%, preferably 20 to 40% by weight of the pharmaceutical composition.
Preferably however no such additive is present or is only present in an amount less than 1% by weight of the pharmaceutical composition.
If desired the pharmaceutical composition may contain a hydrophobic elastomer, e.g. a synthetic resin. Such resins are conventional in the plaster art. Suitable resins may include non-swellable acrylate resins. These may if desired be adhesive. The weight ratio of polymer, e.g. hydrophilic polymer to resin may for example be from 1:0.5 to 1:10. The resin may contain modifiers, extenders, e.g. of softening point about 50 to 100xc2x0 C. Such extenders may have adhesive or softening properties. Examples of such extenders may include resin acids, glyceryl and phthalate esters of resin acids.
A preferred pharmaceutical composition according to the invention comprises
a) (S)-N-ethyl-3-[1-dimethylamino)ethyl]-N-methyl-phenyl-carbamate as compound A in free base form in an amount of 20 to 40 weight-%,
b) polymethacrylate in an amount of 10 to 30% by weight
c) acrylate copolymer in an amount of 40 to 60% by weight, and
d) xcex1-tocopherol in an amount of between 0.05 and 0.3% by weight
wherein the total weight of the pharmaceutical composition is 100%.
In another aspect the present invention provides the use of an anti-oxidant to stabilize a pharmaceutical composition containing Compound A.
Before the finding by the present applicant that an anti-oxidant is necessary in compositions of this invention, it was hitherto thought unnecessary.
The applicant has found that an effective stabilising effect is surprisingly achieved when the antioxidant is selected from tocopherol, esters thereof, e.g. tocopherol acetate, ascorbyl palmitate, ascorbic acid, butylhydroxytoluene, butylhydroxyanisole or propyl gallate, preferably xcex1-tocopherol or ascorbyl palmitate. The antioxidant may be conveniently present in an amount of from about 0.01 to about 0.5%, e.g. 0.05 to 0.20, e.g. 0.15%, more particularly 0.1% by weight based on the total weight of the pharmaceutical composition.
Pharmaceutical compositions of the invention produced in analogous manner to example 1 described hereinafter containing 0.1% tocopherol show for Example only 1.3% degradation products compared to 4.46% degradation products in equivalent compositions not containing tocopherol in 2 month stress tests at 60xc2x0 C. Pharmaceutical compositions of the invention in analogous manner to example 1 described hereinafter containing 0.15% tocopherol show for example only 0.25% degradation products compared to 1.09% degradation products in compositions not containing tocopherol in 3 month stress tests at 40xc2x0 C. at 75% room humidity.
The pharmaceutical composition of the invention is preferably used for transdermal application.
In another aspect of the invention there is provided a transdermal device for administering a Compound A which comprises a pharmaceutical composition containing Compound A, a backing layer providing support for the pharmaceutical composition, an adhesive for fixing the pharmaceutical composition to the backing layer and a release-liner releasably contacting said adhesive.
The pharmaceutical composition may be conveniently contained in a discrete thin layer, the upper and lower surfaces of which may be coated in a layer of adhesive the surface of which in turn provide backing layer and release-liner contacting surfaces.
The pharmaceutical composition contained in the discrete layer may comprise the Compound A and other excipients in a polymer matrix, the polymer matrix therefor being provided by the diluent or carrier aforementioned. If desired Compound A may be dispersed throughout, or dissolved in, said polymer matrix.
The transdermal device may alternatively be of a more simple construction wherein the polymer matrix containing the pharmaceutical composition additionally comprises an adhesive. In such a simple construction there is no need for the layers of the aforementioned adhesive in order to fix and releasably fix respectively the backing layer and release-liner as the polymer matrix containing the Compound A is self adhesive.
The thickness of the pharmaceutical composition layer in a transdermal device may be in the order of from 20 to 100 xcexcm, more preferably 60 to 100.
The backing layer is preferably made of poly(ethylene terephthalate) PET foil. The backing layer should be thick enough to resist wrinkling which may arise upon prolonged periods in storage and through the movement of a subject""s skin. Typically, the backing layer is, e.g. from approximately 10 xcexcm to 15 xcexcm, in thickness.
In a preferred embodiment, the backing layer is a double layer which consists of a PET layer as aforementioned and an EVA layer, e.g. Scotch Pack 1012.
The release-liner may be a disposable element which serves to protect the pharmaceutical composition prior to its application. Typically the release-liner is produced from a material impermeable to compound A, and adhesive. This release-liner may be easily stripped away from the adhesive. A preferred release-liner is made of poly(ethylene terephthalate) PET foil. A release-liner, e.g. of about 50 to 250 xcexcm, e.g. 100 xcexcm thickness PET film, may be applied over the pharmaceutical composition.
The release liner may be silicone-coated. Said coating is preferably formed of any fluorosilicone compound which is conventionally used in the art, e.g a polyfluoroalkylsiloxane.
It is particularly preferred to employ such a fluorosilicone coating when the adhesive used to affix the pharmaceutical composition to the release liner is not itself a silicone adhesive.
The adhesive may be chosen from any adhesive suitable for skin contact and is preferably an adhesive in which Compound A dissolves at least partly. Preferably the adhesive is a contact adhesive which is pressure sensitive. Preferred adhesive are chosen from amine-resistant silicone pressure sensitive adhesives known in the art, for example the BIO-PSA adhesives produced by Dow Coming Corporation, in particular BIO-PSA Q7-4302.
In a very simple construction of the transdermal device, the adhesive may in fact be the polymer of the polymer matrix.
In a further embodiment, the invention provides a transdermal device comprising a backing layer, a layer comprising compound A in a polymer matrix, a release-liner and, disposed between the layer comprising compound A in a polymer matrix and the release liner, a discrete layer of adhesive material for releasably fixing said transdermal device to patients skin.
Preferably, the adhesive material is a silicone adhesive chosen from amine-resistant silicone pressure sensitive adhesives as hereinabove described.
Typically, a transdermal device of said further embodiment comprises:
a) a polymethacrylate backing layer
b) Compound A in free base form in an acrylate copolymer
c) a BIO-PSA Q7-4302 silicone adhesive layer
d) a release-liner.
Preferably, said further embodiment also comprises silicone oil, e.g. silicone oil Q7-9120 from Dow Coming Corporation, in an amount of 0.1 to 5% by weight, e.g. 1%. The backing layer thickness is preferably from 10 to 50 xcexcm, e.g. 23 xcexcm, and has preferably a round shape.
In general transdermal devices of the invention may be produced in a simple manner. A solvent-evaporation process may be used for said compositions. Thus all the ingredients of the pharmaceutical composition may be mixed in a solvent, e.g. acetone, ethylacetate or hexane, and cast onto a substrate which may act as the backing layer or the release-liner.
The transdermal device aforementioned may be conveniently formed in continuous sheets and may be cut into patches of any desirable size or configuration before use. However, the patches so-formed may expose the pharmaceutical composition-containing layer of the laminate to the atmosphere at the outer edges of the patch.
In an alternative embodiment, however, a transdermal device is provided wherein in the patches formed therefrom, the pharmaceutical composition is not exposed to the atmosphere during storage or during application. Such patches further reduce the likelihood of the Compound A being exposed to oxidative influences. The transdermal device may comprise, e.g. a continuous backing layer, a continuous release-liner and located there-between, in discrete portions, a pharmaceutical composition portion, the backing layer being configured such that it may be releasably fixed with an adhesive to the release-liner so to seal said pharmaceutical composition in a pocket defined by the inner surface of the backing layer and inner surface of the release-liner. This embodiment may be conveniently referred to as a cover patch.
The pocket described hereinabove is preferably filled with an adhesive so as to encapsulate completely the discrete portion of pharmaceutical composition. Preferably the adhesive is a silicone pressure sensitive adhesive as described hereinabove.
It is an optional feature of all the transdermal devices described hereinabove that they comprise a layer of adhesive between the pharmaceutical composition and the release liner. This, has the primary function of fixing the release liner in contact with the remainder of the device thus protecting the pharmaceutical composition before use. However, if the adhesive is a silicone adhesive, then the layer may additionally act as a membrane through which the Compound A may pass at a controlled rate into the patient through the skin. Without wishing to be limited to a particular theory, it is suggested that the Compound A, dispersed throughout the polymer matrix exhibits little tendency to migrate into the silicone adhesive layer during storage. Accordingly, there is relatively low concentration of Compound A in the silicone layer. In use, the subjects skin, however, may display a much higher affinity for Compound A than the silicone layer and the initial low concentration of Compound A in the silicone layer passes into the subject""s body. The silicone layer surprisingly prevents the subject from receiving a sudden high dose of Compound A upon application of the device and instead promotes a gradual increase of concentration in the subject.
The cover patch transdermal device may conveniently be formed as a continuous sheet or webbing and may be cut, or tom along a frangible area dividing each device, into patches before use although such devices may be provided as discrete patches.
The transdermal devices of the invention in general have, for example an effective contact area of pharmaceutical composition on the skin of from about 1 to about 80 square centimeters, preferably about 10 square centimeters, and are intended to be applied at intervals of about once every 1 to 7 days, preferably 1-3 days. Compound A is well tolerated at a dose of 36 mg in free base form in up to 80 cm2 of patches according to the invention containing 36 mg compound A from which 12 mg was absorbed. Compound A may, for example be administered at a dose of 8 mg in a patch of ca. 10 cm2, once every day. The patch may be applied, for example on the abdomen, thigh, behind an ear, or on a shoulder or upper arm.
The pharmaceutical composition, optionally formed as a transdermal device, of the present invention are useful for the same indications as for known compositions containing compound A. The exact amounts of compound A to be administered may depend on a number of factors, e.g. the drug release characteristics of the compositions, the drug penetration rate observed in vitro and in vivo tests, the duration of action required, the form of compound A, and for transdermal compositions the size of the skin contact area, and the part of the body to which the unit is fixed. The amount of and, e.g. area of the composition etc. may be determined by routine bioavailability tests comparing the blood levels of active agents after administration of compound A in a composition according to the invention to intact skin and blood levels of Compound A observed after oral administration of a therapeutically effective dose of the compound.
Orally, the Compound A is well tolerated at an initial dose of 1.5 mg twice a day orally and the dose may be stepped up to 3 mg twice daily in week 2. Higher dosages are possible, for example 4.5 mg twice daily and even 6 mg twice daily. Tolerability is seen to be even better for the transdermal device, wherein 24 mg were absorbed in 24 hours.

LTS Lohmann Therapie-Systeme GmbH Co. KG


Aspects of the present invention disclose a method, computer program product, and system for interpreting text entry for an input/output device. The method includes one or more processors receiving input from a set of sensors. The sensors are grouped in segments corresponding to a finger of a user. The method further includes one or more processors identifying input by a user of a gesture into a sensor of the set of sensors. The method further includes one or more sensors determining a first alphanumeric character that corresponds to the identified gesture input by the user. The method further includes one or more processes generating an input stream of alphanumeric characters, the input stream comprising the determined first alphanumeric character that corresponds to the identified gesture and one or more additional alphanumeric characters determined from identified gestures input by the user.

The present invention relates generally to the field of data entry, and more particularly to interpreting text entry into an input/output device.
Text messaging, or texting, is the act of composing and sending electronic messages, typically consisting of alphabetic and numeric characters, between two or more users of mobile phones, fixed devices (e.g., desktop computers), or portable devices (e.g., tablet computers or smartphones). While text messages are usually sent over a phone network, due to the convergence between the telecommunication and broadcasting industries in the 2000s, text messages may also be sent via a cable network or Local Area Network. The term originally referred to messages sent using the Short Message Service (SMS). It has grown beyond alphanumeric text to include multimedia messages (known as MMS) containing digital images, videos, and sound content, as well as ideograms known as emoji (happy faces and other icons).
Text messages are used by youth and adults for personal, family, and social purposes and in business, government, and non-governmental organizations for communication between colleagues. As with e-mailing, the sending of short informal messages has become an accepted part of many cultures. This makes texting a quick and easy way to communicate with friends and colleagues, including in contexts where a phone call would be impolite or inappropriate (e.g., calling very late at night or when one knows the other person is busy with family or work activities). Like e-mail and voice mail, and unlike landline or mobile phone calls (in which the caller hopes to speak directly with the recipient), texting does not require the caller and recipient to both be free at the same moment; this permits communication even between busy individuals. Text messages can also be used to interact with automated systems, for example, to order products or services from e-commerce websites or to participate in online contests. Advertisers and service providers use direct text marketing to send messages to mobile phone users about promotions, payment due dates, and other notifications instead of using postal mail, e-mail, or voicemail.

Interpreting and generating input and output gestures

International Business Machines Corporation


Benzimidazole derivatives and salts and prodrugs thereof are disclosed, together with methods for the treatment of cancers or viral infections in warm-blooded animals by administration of these compounds. Such compounds may be used in combination with a chemotherapeutic agent and/or a potentiator.

The present invention relates to benzimidazole derivatives and their use for the treatment of cancer or a viral infection in warm blooded animals, particularly in humans and other mammals. The methods may use such a compound in combination with a potentiator or a chemotherapeutic agent.
Cancers are a leading cause of death in animals and humans. The exact cause of cancer is not known, but links between certain activities such as smoking or exposure to carcinogens and the incidence of certain types of cancers and tumors has been shown by a number of researchers.
Many types of chemotherapeutic agents have been shown to be effective against cancers and tumor cells, but not all types of cancers and tumors respond to these agents. Unfortunately, many of these agents also destroy normal cells. The exact mechanism for the action of these chemotherapeutic agents are not always known.
Despite advances in the field of cancer treatment the leading therapies to date are surgery, radiation and chemotherapy. Chemotherapeutic approaches are said to fight cancers that are metastasized or ones that are particularly aggressive. Such cytocidal or cytostatic agents work best on cancers with large growth factors, i.e., ones whose cells are rapidly dividing. To date, hormones, in particular estrogen, progesterone and testosterone, and some antibiotics produced by a variety of microbes, alkylating agents, and anti-metabolites form the bulk of therapies available to oncologists. Ideally cytotoxic agents that have specificity for cancer and tumor cells while not affecting normal cells would be extremely desirable. Unfortunately, none have been found and instead agents that target especially rapidly dividing cells (both tumor and normal) have been used.
Clearly, the development of materials that would target cancer cells due to some unique specificity for them would be a breakthrough. Alternatively, materials that were cytotoxic to cancer cells while exerting mild effects on normal cells would be desirable.
Human Immunodeficiency Virus (HIV), the etiological agent for AIDS (acquired immune deficiency syndrome), is a member of the lentiviruses, a subfamily of retroviruses. HIV integrates its genetic information into the genome of the host. Most importantly, HIV infects and invades cells of the immune system; it breaks down the body""s immune system and renders the patient susceptible to opportunistic infections and neoplasms. HIV-1 is cytopathic for T4 lymphocytes, cells of the immune system that express the cell surface differentiation antigen CD4. In addition to CD4+ T cells, the host range of HIV includes cells of the mononuclear phagocytic lineage, including blood monocytes, tissue macrophages, Langerhans cells of the skin and dendritic reticulum cells within lymph nodes.
Precursor cells in the bone marrow are released into the blood in an immature circulating form known as monocytes. Monocytes use the blood strictly as a transport medium. Once they arrive where they""re going to be used, they leave the blood and complete differentiation into macrophages. Cells of the monocyte/macrophage lineage are a major target population for infection with HIV in the body and are thought to provide reservoirs of virus for disseminating infection throughout the body. HIV is also neurotropic, capable of infecting monocytes and macrophages in the central nervous system causing severe neurologic damage. They can interact and fuse with CD4-bearing T cells, causing T cell depletion and thus contributing to the pathogenesis of AIDS.
Progression from HIV infection to AIDS is primarily determined by the effects of HIV on the cells that it infects, including CD4+ T lymphocytes and macrophages. In turn, cell activation, differentiation and proliferation regulate HIV infection and replication in those cells. HIV and other lentiviruses can proliferate in nonproliferating, terminally differentiated macrophages and growth-arrested T lymphocytes. This ability of lentiviruses, including HIV, to replicate in nonproliferating cells, particularly in macrophages, is believed to be unique among retroviruses.
Due to the above-mentioned problems in the art, the present inventors have sought improvements and provide such improvements herein.
Carbendazim, or 2-methoxycarbonylaminobenzimidazole, has been studied as a cancer treatment. See U.S. Pat. No. 5,767,138 issued Jun. 16, 1998 to J. B. Camden. Carbendazim metabolizes in the body through the hydroxylation of the benzene ring, primarily in the 5-position. The metabolite is not as active in the treatment of cancer as the 2-methoxycarbonylaminobenzimidazole. Moreover, this compound is not very soluble. A derivative that maintains the activity of the benzimidazole but is more soluble is highly preferred. It has been found that substituted benzimidazole carbamates, and in particular, those claimed herein are more soluble, yet maintain cytotoxic behavior.
The compounds of the present invention are represented by the following formula A:
wherein,
R is hydrogen, xe2x80x94COOR2 or xe2x80x94CONHR2;
R1 is hydrogen, xe2x80x94COOR3 or xe2x80x94CONER3;
each of R2 and R3 is independently alkyl, haloalkyl, alkenyl, haloalkenyl, cycloalkyl, cycloalkalkyl, heterocycloalkyl, heterocycloalkalkyl, substituted or unsubstituted benzyl, hydroxyalkyl, alkoxyalkyl, poly(alkoxy)alkyl, hydroxyalkoxyalkyl, hydroxypoly(alkoxy)alkyl, haloalkoxyalkyl, halopoly(alkoxy)alkyl, or aminoalkyl;
each of X and Y is independently hydrogen, alkyl, alkenyl, cycloalkyl, haloalkyl, haloalkenyl, bromo, chloro, fluoro, nitro, or amino; and
n is 1, 2 or 3.
The benzyl group may optionally be substituted with one or more nitro, carboxy, hydroxy, alkyl, alkoxy, or halide substituents.
Pharmaceutically acceptable salts of the benzimidazole derivatives of formula A are also included in the present invention. Further included in the invention are the prodrugs of the compounds of formula A.
In one presently preferred embodiment of the invention, the benzimidazole derivatives of the invention are of the formula A-1 or A-2:
and more preferably the compounds are of formula A-1 or A-2 where each of X and Y is hydrogen.
In another presently preferred embodiment of the invention, the benzimidazole derivatives are of the formula A-3:
and more preferably, the compounds are of formula A-3 where X is hydrogen and Y is hydrogen or halogen; the halogen is preferably chloro.
In another presently preferred embodiment of the invention, the benzimidazole derivatives are of the formula A-4:
and more preferably, the compounds are of formula A4 where X is hydrogen and Y is hydrogen or halogen; the halogen is preferably chloro.
Methods are provided by the present invention for treatment of warm blooded animals, and in particular, humans and other mammals, that are affected by cancer or viral infection, the methods comprising administering a therapeutically effective amount of a benzimidazole derivative of formula A, or a salt or a prodrug thereof, to the animal.
The term xe2x80x9calkylxe2x80x9d refers to a fully saturated monovalent hydrocarbon radical of 1 to 12 carbon atoms. It may be straight-chain or branched. Preferred are those alkyl groups containing 1 to 10 carbon atoms, with 2 to 8 carbon atoms particularly preferred.
The term xe2x80x9calkenylxe2x80x9d refers to an unsaturated monovalent hydrocarbon radical of 2 to 12 carbon atoms containing only carbon and hydrogen and having one or more double bonds. It may be straight-chain or branched. Preferred are those alkenyl groups containing 2 to 10 carbon atoms, with 2 to 8 carbon atoms particularly preferred.
The term xe2x80x9calkoxyxe2x80x9d means the group xe2x80x94ORxe2x80x2 wherein Rxe2x80x2 is alkyl. Preferred are alkoxy groups having 1 to 10 carbon atoms, more preferably 2 to 8 carbon atoms.
The term xe2x80x9calkoxyalkylxe2x80x9d refers to an alkoxy group covalently attached to an alkyl group. The alkoxy group contains from 1 to 12, preferably from 1 to 6 carbon atoms. The alkoxy group may be substituted with one or more hydroxyl groups (an xe2x80x9chydroxyalkoxyalkylxe2x80x9d) or with one or more halogen atoms (a xe2x80x9chaloalkoxyalkylxe2x80x9d); preferably the hydroxyl or halogen is on the terminal end of the alkoxyalkyl substituent.
The term xe2x80x9cpoly(alkoxy)alkylxe2x80x9d denotes 2 to 200, preferably 2 to 20, alkoxy groups covalently linked in either a linear or a branched configuration and attached to an alkyl group. Linear poly(alkoxy)alkyl moieties have a structure such as
xe2x80x94CH2)mxe2x80x94Oxe2x80x94(CH2)mxe2x80x94Oxe2x80x94(CH2)mxe2x80x94Oxe2x80x94(CH2)mxe2x80x94 . . . xe2x80x94Oxe2x80x94CmH2m+1, where xe2x80x9cmxe2x80x9d is an integer, the same or different along the length of the chain. Branched moieties have two or more (xe2x80x94Oxe2x80x94(CHt)mxe2x80x94) groups bound to a common third (xe2x80x94Oxe2x80x94(CHt)mxe2x80x94) group, where xe2x80x9ctxe2x80x9d has a value that is independently selected from 0, 1 and 2 for each (CHt)m group. Linear configurations are preferred. The number of repeating (xe2x80x94Oxe2x80x94(CHt)mxe2x80x94) groups within a substituent may be up to 200, preferably from 2 to 20, more preferably from 2 to 7, and most preferably is 2-5. The individual alkoxy groups may be the same or different, and individual alkoxy groups preferably contain from 1 to 6 carbon atoms each, and most preferably from 1 to 3 carbon atoms each. A presently preferred poly(alkoxy)alkyl is xe2x80x94(CH2)yxe2x80x94(OCH2CH2)xxe2x80x94OCH3 or
xe2x80x94CH2)yxe2x80x94OCH2CH2)xxe2x80x94OCH2CH3, where y=1-4 and x=1-100, preferably 1-10, and more preferably, 2-5. The individual alkoxy groups may be substituted with one or more hydroxyl groups (an xe2x80x9chydroxypoly(alkoxy)alkylxe2x80x9d) or with one or more halogen atoms (a xe2x80x9chalopoly(alkoxy)alkylxe2x80x9d); preferably the hydroxyl or halogen is on the terminal end of the poly(alkoxy)alkyl substituent.
xe2x80x9cHeterocycloxe2x80x9d designates a heterocyclic group; that is, a closed-ring structure, usually of either 5 or 6 members, in which one or more of the atoms in the ring is an element other than carbon, such as for example sulfur, nitrogen, or oxygen. A heterocyclic group may be, but is not limited to, pyridine, pyrrole, furan, thiophene, morpholine, and purine, optionally substituted with one or more nitro, carboxy, sulfonic acid, hydroxy, alkyl, alkoxy, or halide substituents.
The term xe2x80x9caminoxe2x80x9d refers to primary amines (xe2x80x94NH2), secondary amines (xe2x80x94NHRxe2x80x2), and tertiary amines (xe2x80x94NRxe2x80x2Rxe2x80x3), where Rxe2x80x2 and Rxe2x80x3 are the same or different substituent groups, such as alkyl, alkenyl, halogen, hydroxy, and the like.
xe2x80x9cIndependentlyxe2x80x9d signifies that two or more of the groups immediately preceding the term are either identical or different; i.e., selection of one from the list following the term does not affect selection of the other(s).
xe2x80x9cSubstitutedxe2x80x9d encompasses both single and multiple substitutions, the latter including multiple substitutions by the same substituent as well as mixtures of different substituents.
The term xe2x80x9coptionalxe2x80x9d or xe2x80x9coptionallyxe2x80x9d means that the subsequently described event or circumstance may or may not occur, and that the description includes instances in which said event or circumstance occurs and instances in which it does not. For example, xe2x80x9coptionally substituted phenylxe2x80x9d means that the phenyl may or may not be substituted and that the description includes both unsubstituted phenyl and phenyl wherein there is substitution. As another example, xe2x80x9coptionallyxe2x80x9d followed by xe2x80x9cconverting the free base to the acid addition saltxe2x80x9d means that such conversion may or may not be carried out in order for the process described to fall within the invention, and the invention includes those processes wherein the free base is converted to the acid addition salt and those processes in which it is not.
As used herein, xe2x80x9ca therapeutically effective amountxe2x80x9d means the concentration or quantity or level of the compound of the present invention that can attain a particular medical end, such as control or destruction of cancer cells, virally-infected cells, or viruses without producing unacceptable toxic symptoms. The term xe2x80x9csafe and effective amountxe2x80x9d refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention. The specific xe2x80x9csafe and effective amountxe2x80x9d will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its salts.
As used herein, a xe2x80x9cpharmaceutical addition saltxe2x80x9d or xe2x80x9cpharmaceutically acceptable saltxe2x80x9d is a salt of the benzimidazole derivative compound with an organic or inorganic acid. These preferred acid addition salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and others known to those of ordinary skill in the art.
As used herein, the term xe2x80x9cprodrugxe2x80x9d refers to a form of a benzimidazole derivative compound that has minimal therapeutic activity until it is converted to its desired biologically active form. A prodrug is a compound having one or more functional groups or carriers covalently bound thereto, which functional groups or carriers are removed from the compound by metabolic processes within the body to form the respective bioactive compound.
As used herein, the term xe2x80x9cmetabolitexe2x80x9d refers to the break-down or end product of a benzimidazole derivative compound or its salt produced by metabolism or biotransformation in the animal or human body; e.g., biotransformation to a more polar molecule such as by oxidation, reduction, or hydrolysis, or to a conjugate (see Goodman and Gilman, xe2x80x9cThe Pharmacological Basis of Therapeuticsxe2x80x9d 8th Ed., Pergamon Press, Gilman et al. (eds.), 1990 for a discussion of biotransformation). As used herein, the metabolite of a benzimidazole derivative compound or its salt may be the biologically active form of the compound in the body. An assay for activity of a metabolite of a benzimidazole derivative of the present invention is known to one of skill in the art in light of the present disclosure, for example, testing for efficacy against a virus in vitro or in vivo.
As used herein, a xe2x80x9csubject in need thereofxe2x80x9d is a warm blooded animal having cancer or having a viral infection.
As used herein, xe2x80x9ccancerxe2x80x9d refers to all types of cancers, or neoplasms or benign or malignant tumors. In one embodiment, those cancers that attack normal healthy blood cells or bone marrow are contemplated by the present invention. Preferred cancers for treatment using methods provided herein include carcinoma, sarcoma, lymphoma, or leukemia. By xe2x80x9ccarcinomaxe2x80x9d is meant a benign or malignant epithelial tumor and includes, but is not limited to, breast carcinoma, prostate carcinoma, non-small cell lung carcinoma, colon carcinoma, CNS carcinoma, melanoma carcinoma, ovarian carcinoma, or renal carcinoma. A preferred host is a human host.
A xe2x80x9cviral infectionxe2x80x9d as used herein means an infection due to a DNA virus or an RNA virus (retrovirus). Examples of a double-stranded DNA virus are the Herpes virus and the influenza virus. Human immunodeficiency virus (HIV) is a prototype for retroviruses, i.e., viruses that use reverse transcription to replicate. However, certain DNA viruses use, in part, reverse transcription mechanisms to replicate such as, for example, the Hepatitus B virus. xe2x80x9cVirusesxe2x80x9d include retroviruses such as HIV or HTLV, influenza, rhinoviruses, herpes, hepatitis, or the like.
As used herein, a benzimidazole derivative of formulas A and A-1 through A-4, or a pharmaceutical salt thereof or a prodrug thereof, are xe2x80x9ccompounds of the present invention.xe2x80x9d Such compounds are further set forth under B infra.
As used herein, xe2x80x9cchemotherapeutic agentsxe2x80x9d includes DNA-interactive agents, antimetabolites, tubulin-interactive agents, hormonal agents and others, such as asparaginase or hydroxyurea, and are as further set forth under D infra.
As used herein, xe2x80x9cpotentiatorsxe2x80x9d are materials that affect the immune system or enhance the effectiveness of a compound of the present invention and are further set forth under E herein.
Following long-standing patent law convention, the terms xe2x80x9caxe2x80x9d and xe2x80x9canxe2x80x9d mean xe2x80x9cone or morexe2x80x9d when used in this application, including the claims.
The benzimidazole derivatives of the present invention are those of formula A, as set forth above. In one embodiment of the invention, presently preferred compounds are those of formulas A-1 and A-2. In another embodiment, presently preferred compounds are those of formulas A-3 and A-4.
Pharmaceutically acceptable salts of the benzimidazole compounds are considered within the scope of compounds of the present invention. They are salts with an organic or inorganic acid. Preferred acid addition salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, or the like. Such salts may be synthesized from the compound of the present invention, or derivative thereof, that contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts may be prepared by reacting a free acid or base form of the compound, or derivative thereof, with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Further suitable salts may be found in Remington: The Science and Practice of Pharmacy, 19th ed., Mack Publishing Company, Easton, Pa., 1995, p.1457.
Pharmaceutically acceptable salts of the compounds of the present invention include conventional non-toxic salts or the quaternary ammonium salts of the compounds or derivatives formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, or the like; and salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, or the like. Preferred acid addition salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, or the like. A presently preferred salt is the hydrochloride salt.
Further included within the scope of the compound, or salts thereof, useful for the present invention are prodrugs thereof. As used herein, a xe2x80x9cprodrugxe2x80x9d is a drug covalently bonded to a carrier wherein release of the drug occurs in vivo when the prodrug is administered to a mammalian subject. Prodrugs of the compounds of the present invention are, prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to yield the desired compound. Prodrugs include compounds wherein hydroxy, amine, or sulfhydryl groups are bonded to any group that, when administered to a mammalian subject, is cleaved to form a free hydroxyl, amino, or sulfhydryl group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, or benzoate derivatives of alcohol or amine functional groups in the compounds of the present invention; phosphate esters, dimethylglycine esters, aminoalkylbenzyl esters, aminoalkyl esters or carboxyalkyl esters of alcohol or phenol functional groups in the compounds of the present invention; or the like.
Synthetic Reaction Parameters
The terms xe2x80x9csolventxe2x80x9d, xe2x80x9cinert organic solventxe2x80x9d and xe2x80x9cinert solventxe2x80x9d mean a solvent that is passive or non-reactive under the conditions of the reaction being described in conjunction therewith (including, for example, benzene, toluene, acetonitrile, tetrahydrofuran (xe2x80x9cTHFxe2x80x9d), dimethylformamide (xe2x80x9cDMFxe2x80x9d), chloroform, methylene chloride (or dichloromethane), diethyl ether, methanol, pyridine, and the like). Unless specified to the contrary, the solvents used in the reactions of the present invention are inert organic solvents.
The reaction temperature can vary widely depending on the reactivity of the reactants. However, the temperature should not be so high as to decompose the reactants or so low as to cause inhibition of the condensation or freezing of the solvent. Unless specified to the contrary, the reactions described herein take place at atmospheric pressure over a temperature range from the temperature of dry ice to about 100xc2x0 C., more preferably from about 10xc2x0 C. to about 50xc2x0 C., and most preferably at xe2x80x9croomxe2x80x9d or xe2x80x9cambientxe2x80x9d temperature (xe2x80x9cRTxe2x80x9d), e.g. about 20xc2x0 C.
Unless otherwise specified, the reaction times and conditions are intended to be approximate.
The time required for the reactions herein will depend to a large extent on the temperature being used and the relative reactivities of the starting materials. Therefore, the reaction time can vary greatly, for example from about five minutes to about two days. Various known techniques such as different types of chromatography, especially thin layer chromatography (xe2x80x9cTLCxe2x80x9d), gas chromatography (xe2x80x9cGCxe2x80x9d), or spectroscopy can be used to follow the progress of the reaction by the disappearance of the starting compound(s).
Isolation and purification of the compounds and intermediates described herein can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography, thick-layer chromatography, centrifugal chromatography, or preparatory HPLC, or a combination of these procedures. Specific illustrations of suitable separation and isolation procedures can be found by reference to the examples hereinbelow. However, other equivalent separation or isolation procedures can, of course, also be used.
Synthesis of Compounds of Formula A
The compounds of the invention are prepared by modification to the benzimidazole nucleus:
In one synthetic process, commercially available 2-aminobenzimidazole (101) can be reacted with a variety of chloroformates or isocyanates in pyridine to form, respectively, carbamates (105) or ureas (103) at the N3 position. In this manner, N3-substituted benzimidazole derivatives of formula A-1 (105) or A-2 (103) with a free 2-amino group are synthesized (Reaction Scheme 1).
By heating the N3-acyl analogs (103, 105) in pyridine, the acyl groups can be induced to migrate to the 2-amino position to give benzimidazole derivatives of formula A-3 or A-4 (205 and 203, respectively) (Reaction Scheme 2).
Where it is desired to synthesize a compound of formula A having a chlorine atom at the 5(6) position, 4-chloro-1,2-phenylenediamine 301 is condensed with cyanogen bromide (Zou, R. et al., J. Med. Chem. 40:811-818 (1997)) to give the corresponding chloro 2-aminobenzimidazole 303. The benzimidazole 303 can then be heated with an isocyanate in pyridine to form the chloro 2-aminourea analogs 305 (Reaction Scheme 3).
In another method for preparing the compounds of formula A-3 or A-4, the commercially-available fungicide methyl 2-aminobenzimidazole carbamate (carbendazim, 401) is treated with an appropriate alcohol or amine with or without added toluene as the solvent in the presence of aluminum isopropoxide catalyst to give new carbamates (403) and ureas (405), respectively (Reaction Scheme 4).
Presently Preferred Embodiments
In one embodiment of the present invention, presently preferred benzimidazole derivatives are those where R1 and X are hydrogen, and Y is hydrogen or chloro and is in the 5(6)-position. Further preferred are those compounds where R1, X and Y are hydrogen and R is selected from those groups listed in Table 1: Solubility is based on a standard measure used in medicinal chemistry, the octanol-water partition coefficient, LogP, Lower Log) values indicate higher aqueous solubility. There are a variety of methods to estimate this value for a proposed structure using computer calculations. The below LogP solubility values were determined using the atom typing method of Ghose, Pritchett et al. (J. Comput. Chem. 9(1):80-90, 1988). Carbendazim has a LogP of 1.302.
In another embodiment of the invention, presently preferred benzimidazole derivatives are those where R is hydrogen, X is hydrogen, and Y is hydrogen or chloro and is in the 5(6)-position. Further preferred are those compounds where R and X are hydrogen, Y is hydrogen or chloro in the 5(6) position, and R1 is selected from those groups listed in Table 2:
* Yxe2x95x90Cl, at the 5(6) position
Screening assays for determining those cancers susceptible to treatment using compounds of the present invention include incubating cell line models representing specific cancers as set forth, for example, by the National Cancer Institute, in the presence and absence of such compounds. Viability of cells may be determined by the MTT assay (Promega Corp., Madison, Wis. 53711), or the SRB (sulforhodamine B) assay (Skehan, et al., JNCI, 82:13,1107,1990). Susceptibility to said compounds exists when viability in the presence of a compound of the present invention is less than viability in the absence of such compound.
Exemplary cell line models representing specific cancers include, but are not limited to, the following:
Non-small cell lung cancer: NCIH23, NCIH324, NCIH522, A549/ATCC, A549(ASC), CALU1, EKVX, NCIH125M, NCIH226, NCIH520, SKMES1, NCIH322M, NCIH358M, NCIH460, NCIH292, HOP62, HOP18, HOP19, HOP92, LXFL 529, SW1573, LXFS 650L, ML1019, ML1076, ML1045, or UABLG22;
Small cell lung cancer: NCIH69, NCIH146, NCIH82, NCIH524, DMS 114, DMS 273, HOP27, SHP77, or RHOS;
Colon cancer: HT29, HCC2998, HCT116, LOVO, SW1116, SW620, COLO 205, DLD1, WIDR, COLO 320DM, HCT15, CXF 280, KM12, KM20L2, COLO 741, CXF 264L, COLO 746, UABC02, MLI059, CAC02, HT29/PAR, HT29/MDR1, or NB4;
Breast cancer: MCF7, MCF7/ADRRES, ZR751, ZR7530, MDAMB231/ATCC, HS 578T, UISOBCA1, MCF7/ATCC, SKBR3, MDAMB435, MDAN, BT549, T47D, MDAMB231, MAXF 401, BT474, or MDAMB468;
Ovarian cancer: OVCAR3, OVCAR4, OVCAR5, OVCAR8, A2780, IGROV1, SKOV3, OVXF 899, A1336, or ES2;
Leukemia: P388, P3888/ADR, CCRFCEM, CCRFSB, K562, MOLT4, L1210, HL60(TB), RPM18226, SR, or K562/ADR;
Fibroblast: IMR90, or CCD19LU;
Renal cancer: UO31, SN12C, SN12S1, SN12K1, SN12L1, SN12A1, A498, A704, CAKI1, RXF 393, RXF631, 7860, SW156, TK164, 769P, SS78, ACHN, TK10, RXF 486L, UOK57, or UOK57LN;
Melanoma: LOX IMVI, MALME3M, RPMI7951, SKMEL2, SKMEL5, SKMEL28, SKMEL31, UCSD 242L, UCSD 354L, M14, M19MEL, UACC62, UACC257, MEXF 514L, or UABMEL3;
Prostate cancer: PC3, PC3M, DU145, LNCAP, 1013L, UMSCP1, WIS, JE, RER, MRM, DHM, AG, RB, RVP, FC, WAE, DB/SMC, JCA1, ND1, WMF, TSUPRI, JECA, GDP, T10, WBW, RVP1, or WLL;
CNS cancer: SNB7, SNB19, SNB44, SNB56, SNB75, SNB78, U251, TE671, SF268, SF295, SF539, XF 498, SW 1088, SW 1783, U87 MG, SF767, SF763, A172, or SMSKCNY;
Bone/muscle: A204/ATCC, OHS, TE85, A673, CHA59, MHM 25, RH18, RH30, or RD; and
Lymphoma: AS283, HT, KD488, PA682, SUDHL7, RL, DB, SUDHL1, SUDHL4, SUDHL10, NUDUL1, or HUT 102.
Chemotherapeutic agents are generally grouped as DNA-interactive agents, antimetabolites, tubulin-interactive agents, hormonal agents, other agents such as asparaginase or hydroxyurea, and agents as set forth in Table 3. Each of the groups of chemotherapeutic agents can be further divided by type of activity or compound. Chemotherapeutic agents used in combination with a compound of the present invention, or salts thereof of the present invention may be selected from any of these groups but are not limited thereto. For a detailed discussion of the chemotherapeutic agents and their method of administration, see Dorr, et al, Cancer Chemotherapy Handbook, 2d edition, pages 15-34, Appleton and Lang (Connecticut, 1994) herein incorporated by reference.
DNA-interactive agents include alkylating agents, e.g. cisplatin, cyclophosphamide, altretamine; DNA strand-breakage agents, such as bleomycin; intercalating topoisomerase II inhibitors, e.g., dactinomycin and doxorubicin); nonintercalating topoisomerase II inhibitors such as, etoposide and teniposide; and the DNA minor groove binder plicamydin, for example.
The alkylating agents form covalent chemical adducts with cellular DNA, RNA, or protein molecules, or with smaller amino acids, glutathione, or similar chemicals. Generally, alkylating agents react with a nucleophilic atom in a cellular constituent, such as an amino, carboxyl, phosphate, or sulfhydryl group in nucleic acids, proteins, amino acids, or in glutathione. The mechanism and the role of these alkylating agents in cancer therapy is not well understood.
Typical alkylating agents include, but are not limited to, nitrogen mustards, such as chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, uracil mustard; aziridine such as thiotepa; methanesulphonate esters such as busulfan; nitroso ureas, such as carmustine, lomustine, streptozocin; platinum complexes, such as cisplatin, carboplatin; bioreductive alkylator, such as mitomycin, and procarbazine, dacarbazine and altretamine.
DNA strand breaking agents include bleomycin, for example.
DNA topoisomerase II inhibitors include the following intercalators, such as amsacrine, dactinomycin, daunorubicin, doxorubicin (adriamycin), idarubicin, and mitoxantrone; nonintercalators, such as etoposide and teniposide, for example.
A DNA minor groove binder is plicamycin, for example.
Antimetabolites interfere with the production of nucleic acids by one of two major mechanisms. Certain drugs inhibit production of deoxyribonucleoside triphosphates that are the immediate precursors for DNA synthesis, thus inhibiting DNA replication. Certain of the compounds are analogues of purines or pyrimidines and are incorporated in anabolic nucleotide pathways. These analogues are then substituted into DNA or RNA instead of their normal counterparts.
Antimetabolites useful herein include, but are not limited to, folate antagonists such as methotrexate and trimetrexate; pyrimidine antagonists, such as fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, and floxuridine; purine antagonists include mercaptopurine, 6-thioguanine, fludarabine, pentostatin; and ribonucleotide reductase inhibitors include hydroxyurea.
Tubulin interactive agents act by binding to specific sites on tubulin, a protein that polymerizes to form cellular microtubules. Microtubules are critical cell structure units. When the interactive agents bind the protein, the cell can not form microtubules. Tubulin interactive agents include vincristine and vinblastine, both alkaloids and paclitaxel (Taxol), for example.
Hormonal agents are also useful in the treatment of cancers and tumors. They are used in hormonally susceptible tumors and are usually derived from natural sources. Hormonal agents include, but are not limited to, estrogens, conjugated estrogens and ethinyl estradiol and diethylstilbesterol, chlortrianisen and idenestrol; progestins such as hydroxyprogesterone caproate, medroxyprogesterone, and megestrol; and androgens such as testosterone, testosterone propionate; fluoxymesterone, and methyltestosterone.
Adrenal corticosteroids are derived from natural adrenal cortisol or hydrocortisone. They are used because of their anti-inflammatory benefits as well as the ability of some to inhibit mitotic divisions and to halt DNA synthesis. These compounds include, but are not limited to, prednisone, dexamethasone, methylprednisolone, and prednisolone.
Leutinizing hormone releasing hormone agents or gonadotropin-releasing hormone antagonists are used primarily the treatment of prostate cancer. These include leuprolide acetate and goserelin acetate. They prevent the biosynthesis of steroids in the testes.
Antihormonal antigens include, for example, antiestrogenic agents such as tamoxifen, antiandrogen agents such as flutamide; and antiadrenal agents such as mitotane and aminoglutethimide.
Further agents include the following: hydroxyurea appears to act primarily through inhibition of the enzyme ribonucleotide reductase, and asparaginase is an enzyme which converts asparagine to nonfunctional aspartic acid and thus blocks protein synthesis in the tumor.
Taxol (paclitaxel) is a preferred chemotherapeutic agent.
A listing of currently available chemotherapeutic agents according to class, and including diseases for which the agents are indicated, is provided as Table 3.
A xe2x80x9cpotentiator,xe2x80x9d as used herein, is a material that improves or increases the efficacy of the benzimidazole derivatives or a salt or a prodrug thereof, or that acts on the immune system as an immunomodulator. Potentiators can be used in combination with a compound of the present invention. A potentiator may be an antiviral agent. One such potentiator is triprolidine or its cis-isomer. Triprolidine is described in U.S. Pat. No. 5,114,951 (1992, the patent is incorporated by reference herein). A further potentiator is procodazole, (also named 1H-benzimidazole-2-propanoic acid, or xcex2-(2-benzimidazole) propionic acid or 2-(2-carboxyethyl)benzimidazole or propazol). Procodazole is a non-specific immunoprotective agent active against viral and bacterial infections and may be used in combination with the compounds set forth herein. Procodazole is effective with a compound of the present invention, alone in treating cancers, tumors, leukemia or viral infections, or combined with a chemotherapeutic agent.
Further potentiators include, but are not limited to, propionic acid, salts thereof, or esters thereof; antioxidant vitamins such as vitamins A, C, E, or beta-carotene; abacavir; AL-721 (lipid mixture); amprenavir; Amphotericin B methyl ester; Ampligen (mismatched RNA); anti-AIDS antibody; anti-human interferon-xcex1 antibody; anti-AIDS antibody, ascorbic acid and derivatives thereof; AS-101 (heavy metal based immunostimulant); azidothymidine; xcex2-interferon; Bropirimine; butylated hydroxytoluene; Ciamexon, Cimetidine; CL-246,738, colony stimulating factors, including GM-CSF; Creme Pharmatex (benzalkonium chloride); CS-82 (5-unsubstituted derivative of Zidovudine); Cyclosporin; D-penicillamine (3-mercapto-D-valine); delavirdine; dextran sulphate; dinitrochlorobenzene; efavirenz; erythropoietin; Foscarnet (trisodium phosphonoformate); fusidic acid; ganciclovir; glucan; glycyrrhizin, HPA-23 (ammonium-21-tungsto-9-antimonate); human immunevirus antiviral; hyperimmune gamma-globulin, IMREG-1, IMREG-2; indinavir; interferon-xcex1; interferon-gamma; interleukin-1 or interleukin-2; isoprinosine; Krestin; LC-9018; lamivudine; lentilart; LF-1695; methionine-enkephalin; Minophagen C; muramyl tripeptide; naltrexone; nelfinavir; Neutropin; nevirapine; Nonoxinol; Ornidyl (eflornithine); non-nucleoside inhibitors of reverse transcriptase; nucleoside analogues (ddA, ddC, ddI, ddT, ddG, AZT, and the like); pentamidine isethionate; Phenytoin; polymannoacetate; Peptide T (octapeptide sequence); protease inhibitors; Ribavirin; Rifabutin (ansamycin); ritonavir; RNA immunomodulator; rsT4 (recombinant soluble T4); saquinavir; shosaikoto and ginseng; SK-818 (germanium-derived antiviral); sodium diethylthiocarbarmate; stavudine; stearic acid derivative; suramin and analogues thereof; thymic humoral factor; TP-5; Thymosin fraction 5 and Thymosin 1; Thymostimulin; TNF (tumor necrosis factor), vitamin B preparations; Trimetrexate; UA001; alpha-interferon; or acyclovir, for example.
A compound, or a salt or a prodrug thereof, of the present invention may be combined with a potentiator and with a chemotherapeutic agent in the methods of the present invention.
Any suitable dosage may be administered in the methods of the present invention. The compound or salt or prodrug thereof chosen for a particular application, the carrier and the amount will vary widely depending on the species of the warm blooded animal or human, the type of cancer, or the particular viral infection being treated, and depending upon the effective inhibitory concentrations observed in trial studies. The dosage administered will, of course, vary depending upon known factors, such as the pharmacodynamic characteristics of the particular compound, salt, or combination and its mode and route of administration; the age, health, or weight of the subject; the nature and extent of symptoms; the metabolic characteristics of the drug and patient, the kind of concurrent treatment; the frequency of treatment; or the effect desired.
Generally a dosage of as little as about 1-2 milligram (mg) per kilogram (kg) of body weight is suitable, but preferably as little as 10 mg/kg and up to about 10,000 mg/kg may be used. Preferably, a dosage from 15 mg/kg to about 5000 mg/kg is used. Most preferably, the dose is between 150 mg/kg to about 1000 mg/kg. Doses useful in the treatment of cancer or viral infections are 250 mg/kg, 500 mg/kg, 800 mg/kg, 1000 mg/kg, 1500 mg/kg, 2500 mg/kg, 3500 mg/kg, 4000 mg/kg, 5000 mg/kg, or 6000 mg/kg. Any range of doses can be used. Generally, a compound, salt thereof, prodrug thereof, or combination of the present invention can be administered on a daily basis one or more times a day, or one to four times a week, either in a single dose or separate doses during the day. Twice-weekly dosing over a period of at least several weeks is preferred, and often dosing will be continued over extended periods of time and possibly for the lifetime of the patient. However, the dosage and the dosage regimen will vary depending on the ability of the patient to sustain the desired and effective plasma levels of the compounds of the present invention, or salt or prodrug thereof, in the blood.
The compound, salt thereof, prodrug thereof, or combination may be micronized or powdered so that it is more easily dispersed and solubilized by the body. Processes for grinding or pulverizing drugs are well known in the art. For example, a hammer mill or similar milling device can be used. The preferred particle size is less than about 100xcexc and preferably less than 50xcexc.
Intravenously, the most preferred doses may range from about 1 to about 10 mg/kg/minute during a constant rate infusion.
The dosage for humans is generally less than that used in mice and is typically about {fraction (1/12)} of the dose that is effective in mice. Thus, if 500 mg/kg was effective in mice, a dose of 42 mg/kg would be used in humans. For a 60 kg man, this dose would be 2520 mg.
The compounds and salts and prodrugs thereof of the present invention are generally safe. The LD50 is high, about 1500 mg/kg given orally in mice and there are no special handling requirements. The compounds can be given orally, and since they are not very soluble, they are preferably given in tablet form or as a suspension. Alternatively, when micronized to sufficiently small size, they may be and preferably are given as an injection.
The compounds and salts and prodrugs thereof of the present invention may be administered in a unit dosage form which may be prepared by any methods known to one of skill in the art in light of the present disclosure. Unit dosages may include from 1 milligram to 1000 milligrams of active ingredient. Preferably the dosage unit will contain from about 10 mg to about 500 mg active ingredient. The active ingredient is generally present in an amount of about 0.5% to about 95% by weight based on the total weight of the dosage unit.
For intravenous use, preferred dosages may range from about 1 to about 10 mg/kg/minute during a constant rate infusion.
A dosage unit may comprise a single compound, or mixtures thereof, with other compounds or other cancer- or viral-inhibiting compounds. The dosage unit may comprise diluents, extenders, carriers, liposomes, or the like. The unit may be in solid or gel form such as pills, tablets, capsules and the like or in liquid form suitable for oral, rectal, topical, intravenous injection or parenteral administration or injection into or around the treatment site. The range and ratio of benzimidazole derivative, or salt or prodrug thereof, to chemotherapeutic agent or to potentiator will depend on the type of cancer or viral infection being treated and the particular chemotherapeutic agent or potentiator.
Formulations of the present invention include the compound of the present invention, a salt thereof or a prodrug thereof and, optionally, a chemotherapeutic agent and, optionally, a potentiator, generally mixed with a pharmaceutically acceptable carrier. A xe2x80x9cpharmaceutical carrierxe2x80x9d is a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering a compound of the present invention to the animal or human. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. A xe2x80x9cpharmaceutically acceptablexe2x80x9d component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.
Oral formulations suitable for use in the practice of the present invention include capsules, gels, cachets, tablets, effervescent or non-effervescent powders or tablets, powders or granules; as a solution or suspension in aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil emulsion. The compounds of the present invention may also be presented as a bolus, electuary, or paste.
Generally, formulations are prepared by uniformly mixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. A pharmaceutical carrier is selected on the basis of the chosen route of administration and standard pharmaceutical practice. Each carrier must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. This carrier can be a solid or liquid and the type is generally chosen based on the type of administration being used. Examples of suitable solid carriers include lactose, sucrose, gelatin, agar and bulk powders. Examples of suitable liquid carriers include water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions, and solution and or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid carriers may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Preferred carriers are edible oils, for example, corn or canola oils. Polyethylene glycols, e.g. PEG, are also preferred carriers.
The formulations for oral administration may comprise a non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, cyclodextrin, cyclodextrin derivatives, or the like.
Capsule or tablets can be easily formulated and can be made easy to swallow or chew. Tablets may contain suitable carriers, binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, or melting agents. A tablet may be made by compression or molding, optionally with one or more additional ingredients. Compressed tables may be prepared by compressing the active ingredient in a free flowing form (e.g., powder, granules) optionally mixed with a binder (e.g., gelatin, hydroxypropylmethylcellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked carboxymethyl cellulose) surface-active or dispersing agent. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, or the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, or the like. Disintegrators include, for example, starch, methyl cellulose, agar, bentonite, xanthan gum, or the like. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.
The tablets may optionally be coated or scored and may be formulated so as to provide slow- or controlled-release of the active ingredient. Tablets may also optionally be provided with an enteric coating to provide release in parts of the gut other than the stomach.
Exemplary pharmaceutically acceptable carriers and excipients that may be used to formulate oral dosage forms of the present invention are described in U.S. Pat. No. 3,903,297 to Robert, issued Sep. 2, 1975, incorporated by reference herein. Techniques and compositions for making dosage forms useful in the present invention are described in the following references: 7 Modem Pharmaceutics, Chapters 9 and 10 (Banker and Rhodes, Editors, 1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976).
Formulations suitable for topical administration in the mouth wherein the active ingredient is dissolved or suspended in a suitable carrier include lozenges which may comprise the active ingredient in a flavored carrier, usually sucrose and acacia or tragacanth; gelatin, glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
Topical applications for administration according to the method of the present invention include ointments, cream, suspensions, lotions, powder, solutions, pastes, gels, spray, aerosol or oil. Alternately, a formulation may comprise a transdermal patch or dressing such as a bandage impregnated with an active ingredient and optionally one or more carriers or diluents. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
The topical formulations may desirably include a compound that enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
The oil phase of an emulsion used to treat subjects in the present invention may be constituted from ingredients known to one of skill in the art in light of the present disclosure. An emulsion may comprise one or more emulsifiers. For example, an oily phase may comprise at least one emulsifier with a fat or an oil, with both a fat and an oil, or a hydrophilic emulsifier may be included together with a lipophilic emulsifier that acts as a stabilizer. Together, the emulsifier(s), with or without stabilizer(s), make up an emulsifying wax, and the wax together with the oil and/or fat make up the emulsifying ointment base that forms the oily dispersed phase of the cream formulations.
Emulsifiers and emulsion stabilizers suitable for use in the formulation include Tween 60, Span 80, cetosteryl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate, paraffin, straight or branched chain, mono-or dibasic alkyl esters, mineral oil. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, the properties required and compatibility with the active ingredient.
Compounds of the present invention may also be administered vaginally, for example, as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing appropriate carriers in addition to the active ingredient. Such carriers are known in the art in light of the present disclosure.
Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
Formulations suitable for nasal administration may be administered in a liquid form, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer, including aqueous or oily solutions of the active ingredient. Formulations for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, of less than about 100 microns, preferably less than about 50 microns, which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
Formulations suitable for parenteral administration include aqueous and non-aqueous formulations isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending systems designed to target the compound to blood components or one or more organs. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules or vials. Extemporaneous injections solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
In general, water, a suitable oil, saline, aqueous dextrose (glucose), or related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents and, if necessary, buffer substances. Antioxidizing agents, such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid salts thereof, or sodium EDTA. In addition, parenteral solutions may contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, or chlorobutanol. Suitable pharmaceutical carriers are described in Remington, cited supra.
The present invention additionally contemplates administering compounds of the herein described invention for use in the form of veterinary formulations, which may be prepared, for example, by methods that are conventional in the art in light of the present disclosure.
Useful pharmaceutical dosage formulations for administration of the compounds of the present invention are illustrated as follows:
Capsules: A large number of unit capsules are prepared by filling standard two-piece hard gelatin capsules each with 100 milligrams of powdered active ingredient, 150 milligrams of lactose, 50 milligrams of cellulose, and 6 milligrams magnesium stearate.
Soft Gelatin Capsules: A mixture of active ingredient in a digestible oil such as soybean oil, cottonseed oil or olive oil is prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 100 milligrams of the active ingredient. The capsules are washed and dried.
Tablets: A large number of tablets are prepared by conventional procedures so that the dosage unit was 100 milligrams of active ingredient, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275 milligrams of microcrystalline cellulose, 11 milligrams of starch and 98.8 milligrams of lactose. Appropriate coatings can be applied to increase palatability or delay absorption.
Injectable: A parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredient in 10% by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized.
Suspension: An aqueous suspension is prepared for oral administration so that each 5 ml contains 100 mg of finely divided active ingredient, 200 mg of sodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution, U.S.P., and 0.025 ml of vanillin.
Compounds of the present invention may be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.
Compounds of the present invention may be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention can be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.
The method of treatment can be any suitable method which is effective in the treatment of the particular cancer or viral infection that is being treated. Treatment includes administering a therapeutically effective amount of the compounds of the present invention in a form described hereinabove, to a subject in need of treatment.
Compounds of the present invention can be administered by any means that produces contact of the active agent with the agent""s site of action in the body, for example, suitable means including, but not limited to, oral, rectal, nasal, topical (including transdermal, aerosol, buccal or sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous or intradermal), intravesical, or injection into or around the cancer or site of viral infection. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutics. Preferably, compounds of the present invention are administered as a pharmaceutical formulation comprising at least one compound of the present invention, as defined above, together with one or more pharmaceutically acceptable carriers. It can be co-administered in the form of a tablet or capsule, as an agglomerated powder, or in a liquid form, or as a liposome.
The preferred route will vary with the condition and age of the recipient, virus or cancer being treated nature of the disorder, or severity of disorder. It is believed that oral administration, or parenteral treatment is the preferred method of administering the compounds to subjects in need thereof.
In each of the above-described methods, the administering may be in vivo, or may be ex vivo. In vivo treatment is useful for treating diseases in a mammal, preferably the mammal is a human; and ex vivo treatment is useful for purging body fluids, such as blood, plasma, bone marrow, and the like, for return to the body. The nation""s blood supply is currently tested for antibodies to HIV. However, the test is still imperfect and samples that yield negative tests can still contain HIV virus. Treating blood and blood products with the compounds of the present invention can add an extra margin of safety to kill any retrovirus that may have gone undetected. Body tissue may be internal or external to an animal body, or, for example, may be the surface skin of the animal.
Compounds of the present invention may additionally be combined with chemotherapeutic agents and/or potentiators to provide a combination therapy. Combination therapy is intended to include any chemically compatible combination of a compound of the present invention with other compounds of the present invention or other compounds outside of the present invention, as long as the combination does not eliminate the activity of the compound of the present invention. For example, one or more compounds may be combined with a potentiator or with a chemotherapeutic agent. In the case of retroviral infection, a combination therapy with nucleoside analogues such as AZT, nonnucleoside reverse transcriptase inhibitors, TC-3, or protease inhibitors is contemplated by the present invention. In the case of hepatitis, acyclovir, famciclovir or valacyclovir, Ribavirin, interferon, or combinations of Ribavirin and interferon or beta globulin is contemplated as a combination therapy. For herpes, a recombinant alpha interferon can be used as a combination therapy. The active agent can be coadministered, for example, in the form of a tablet or capsule, liposome, as an agglomerated powder, or in a liquid form. The amount of chemotherapeutic agent or potentiator used can be lower than that of the benzimidazole derivative. It will be present in a dosage unit in an amount that provides an operative combination with the benzimidazole derivative. The dosage of the chemotherapeutic agent or the potentiator can range from about 0.5 mg/kg body weight to about 400 mg/kg body weight.
Combination therapy can be sequential, that is the treatment with one agent first and then the second agent, or it can be treatment with both agents at the same time. The sequential therapy can be within a reasonable time after the completion of the first therapy before beginning the second therapy. The treatment with both agents at the same time can be in the same daily dose or in separate doses. For example, treatment with one agent on day 1 and the other on day 2. The exact regimen will depend on the disorder being treated, the severity of the disorder, and the response to the treatment.
In addition to the use of chemotherapeutic agents and potentiators, a benzimidazole derivate or a salt or a prodrug thereof can be combined with a fungicide or an herbicide. Preferred herbicides and fungicides include carbendazim, fluoconazole, benomyl, glyphosate, and propicodazole.
The present invention also includes pharmaceutical kits useful, for example, for the treatment of cancer or viral infection. The kits comprise one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention. Such kits can further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. Instruction, such as printed instructions for example, either as inserts or as labels, the instruction indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.
The following studies were performed to test the effectiveness of the benzimidazole derivatives of the present invention against certain cancers and viral infections.
Colon and Melanoma Tumor Cells Test:
The following cell culture tests were performed to test the toxicity of benzimidazole derivative compounds of the present invention on colon and melanoma tumor cells. The viability of the cells were tested by looking at MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction. MTT assay is a well known measure of cell viability.
The colon tumor cells (HT29) and the melanoma cells (B16 murine melanoma) were seeded (1000-2000 cells) into each well of a 96-well microtiter plate and allowed to grow. Twenty-four hours later, increasing concentrations of each benzimidazole derivative were added to the plates and the cells were allowed to incubate for 3-6 days in the presence of the drugs. MTT reagent was then added to the wells and allowed to incubate for 4 hours at 37xc2x0 C., after which the formazan metabolite was solubilized with acidic isopropanol and the absorbance was read. Experimental controls included blank wells to which no cells were added (zero point of formazan absorbance) and control wells to which no drug was added (highest level of formazan absorbance). Each drug concentration was tested in duplicate and the resulting average absorbance was plotted against drug concentration using the EZ-ED50 computer program on a personal computer interfaced to the microtiter plate reader. The program fit the resulting curves to a four-parameter equation:   Y  =                              A                      ma                                      xe2x80x83                                    x                          -                  A                      m                                      xe2x80x83                                    i                                      xe2x80x83                                    n                                                (                      1            +                          X              /              IC50                                )                n              +          A              m                          xe2x80x83                        i                          xe2x80x83                        n
where Amax and Amin are, respectively, the absorbances in the absence and presence of highest drug concentration, X is the drug concentration and Y is the absorbance at that concentration, n is the slope of the curve, and IC50 is the concentration of drug that gives 50% growth inhibition. The IC50 value is then derived from this equation by EZ-ED50. The IC50 values for growth inhibition of both B16 and HT29 cancer cell lines are reported in Table 4 below (Y=H unless otherwise indicated).
Microtubule Inhibition Assay:
The benzimidazole derivatives of this invention were evaluated for their ability to inhibit formation of microtubules, following the procedure of Luduena, R. F. and M. C. Roach (Pharnacol. Ther. 49: 133-152 (1991)). Tubulin was purified from bovine brain by a cycle of assembly and disassembly, followed by phosphocellulose chromatography and gel filtration (Fellous, A. et al., Eur. J. Biochem. 78(1): 167-174 (1977)). The assay was performed by preparing a sample of tubulin protein under conditions which promote polymerization into microtubules (Prasad, V. et al., J. Protein Chem. 11(5): 509-515 (1992)), then adding the benzimidazole derivatives, at 2 xcexcM, and following the time course of polymerization. Microtubule polymerization was followed by performing the reaction in a cuvette and monitoring UV absorbance at 350 nm, allowing direct visualization of the time course of the polymerization. After 30 minutes, polymerization was complete and the percent inhibition of polymerization was calculated as the final absorbance of the reaction taken as a percentage of the final absorbance of a control polymerization reaction run in parallel without drug added. These data are reported in Table 5 below.
DNA Binding Assay:
The DNA binding assay was performed in a manner very similar to that of the MTT cell growth inhibition assay above. The assay was based upon the ability of the test compounds to displace methyl green (Sigma) from DNA and the resultant hydration of the free methyl green molecule to a colorless derivative (Burres, N. S. et al., J. Nat. Prod. 55(11): 1582-1587 (1992); Kim and Norden, FEBS Lett. 315(1): 61-64 (1993)). As for the MTT assay, each drug concentration was assayed in duplicate and the data were averaged before analysis. Experimental controls in this assay included wells containing free methyl green solution with no DNA, for fully decolorized background absorbance, and methyl green-DNA complex (Sigma) without drug, for the highest possible methyl green absorbance. Methyl green-DNA in buffer was added to each well to obtain A655 of approximately 0.7. Serial dilutions of drug in buffer were added and the plates were mixed, then allowed to stand at room temperature for 24 hours to allow for displacement and hydration of the free methyl green. The absorbance at 655 nm was measured as a function of added drug concentration in an automated microplate reader, and the data were reduced by the computer program EZ-ED50 to derive IC50 values (concentration at which 50% of the methyl green is displaced from DNA).
None of the benzimidazole derivatives listed in Table 4 or 5 showed any DNA interaction by this assay.

Compounds and methods for use thereof in the treatment of cancer or viral infections